Lab Practical 2

In the disinfectant basin

Disposed in disinfectant basin

- notify your instructor

- don’t attempt to pick up broken glass due to cuts happening

- place in a broken glass bin by door of class room

Remove gloves inside out and place in autoclave bags at end of tables.

Dispose in regular trash bin

In trash cans on floor at end of table.

In autoclave bags at end of each table.

In autoclave bags at end of table.

Ocular

Allows to view

Objective

Magnifies what’s on the slide

Condenser

Concentrate the light beam onto the specimen

Course focus knob

Allows for a clear view

Iris diaphragm

Adjust the light of the condenser

Mechanical stage

Holds the slide

Mechanical stage (x-y - stage) knobs

Will adjust the stage

Condenser height adjustment knob

Adjust brightness of light

Condenser centering screws

Rheostat

Base diaphragm

Fine focus knob

Allows for clear viewing

Stage clip

Hold slide still for viewing

1- magnifies

2-

Magnification= ocular * objective

1- magnification

2- resolving power

Clean ocular with q-tip and alcohol

Make sure stage is clean and no slide is left on it

Roll up cord to plug in microscope

make sure stage is lowered

Have the 4X in first position

After setting up microscope place slide on stage with stage clip securing it. Start at 4X objective. Use course focus knob and fine focus knob to view. Then you can go higher on the objective.

Used to increase resolution Power of a microscope.

1- wet hands with soap and water and lather

2- wash entire hands front, back and underneath fingernails

3- using a rotational movement wash both wrist

4- make sure handwashing is 20 seconds

5- Rinse hands

6- thoroughly dry your hands were disposable paper towel

7- turn off the Fossett using the paper towel

80% of infections are transmitted by hands

When hands are visibly soiled

Soap and water

Present, appearing, or found everywhere

A culture that contains a Single species of microbe

When each cell reproduces many times and gives rise

Cloudiness which indicates growth

Nutrient material stable for cultivation of microorganisms

Sampling a pure culture to a new sterile media

Using practice and procedures to prevent contamination

So you don’t kill the bacteria you’re trying to obtain

To help prevent spills and it’s easier to obtain your bacterial sample

If you use too large of a sample you will not have a significant area on the plate to achieve the dilution needed for separation of one cell from another

obtain sample on sterile Q-tip from broth. Lift the lid on the petri plate and swab largest section of the agar. Dispose of swap and autoclave bag. Use loop to complete rest of the procedure. Sterilize your loop. Place the loop in center of first section and gently drag the loop one time into the second section. Lately drag the tip of the loop from side to side and a back-and-forth motion to fill the second section. In between sections sterilize loop. Place loop in center of the second section and gently drag loop one time into third section lightly drag the tip of the loop from side to side in a back-and-forth motion to spread the bacteria to fill the third section. Place the plate lid and sterilize the loop. Stack Algar plates upside down in the class tray to be incubated.

If labeled on top of lid we will not know sections and zones of where we started the bacteria. They are inverted due to it being easier to pick up plates instead of lid being on top because it will spill

Form, evaluation, margin, size, or color

Crystal violet, grams Iodine, acetone, safranin

Crystal violet- primary stain

Grams iodine- mordant that combines with crystal violet in the cell

Acetone- The decolorizer

Safranin - counter stain for secondary stain

Will dissolve the lipids in the outer membrane of gram-negative cell walls

Simple stain is when only one dye is used

Differential stain is when 2 dyes are used

Mycobacterium

Novartis Asteroids- pulmonary disease

Cryptosporidium- diarrheal disease

Mycobacterium- Hansen’s disease

Mycobacterium- TB

Carbon fuchsin is lipid soluble and contains phenol which helps the stain penetrate the cell wall

Like mycobacterium contain large amounts of lipid substance within their cell walls called mycolic acids. These acids resist standing by ordinary methods such as Gram stain

Gram stain uses acetone alcohol (3-5 seconds)

Acid fast uses acid-alcohol decolorizer (15-30 seconds)

Carbolfuchsin dye

Acid-alcohol decolorizer

Methylene blue counterstain

Bacillus anthracis- anthrax

Clostridium botulinum- botulism

Clostridium perfringens- gas gangrene

Clostridium tatami- tetanus

Clostridium difficult- toxic enterocolitis and pseudomembranous colitis

Bacillus anthracis- anthrax

Clostridium botulinum- botulism

Clostridium perfringens- gas gangrene

Clostridium tatami- tetanus

Clostridium difficult- toxic enterocolitis and pseudomembranous colitis

Prepare slide and allow slide to air dry then place the slide on a warmer.

while slide is on warmer put a small piece of film paper on top of the slide and add malachite green to paper.

Keep adding dye as it starts to dry out after five minutes on warmer transfer slide to stain rack for another five minutes

remove filter paper from the side

rinse the slide with Deionized water shake off excess water

add Gram safranin to the slide for two minutes rinse the smear with the ionized water and blot dry with bibulous paper or paper towel observe

then observe results with oil immersion lens of microscope

Positive stain is purple

Negative stain is pink

The negative stain will stain everything except what you want to see. Thus the organism will stand out as a transparent or refractive structure against a contrastingly dark background

BLA gene

GFP

Ara-C

Beta- lactamase

Repressor

Fluorescent protein

Are E. coli cells that have been specially treated to transform efficiently

Heat shock

Electroporation

The repressor protein and will not be transcribed without the addition of the sugar Arabinose to the cells nutrient medium

1 Lucia agar plate with ampicillin and arabinose - prediction of glow is yes

2 luria agar plates with ampicillin- prediction ofof glow is no

1 plain luria agar plate- Prediction of glow is no

Kirby-Bauer- Characteristics as the stability of the antibiotic, the rate of diffusion of the antibiotic, the bacteria being tested the pH of the culture medium, the depth of the culture medium, the inoculum density, The incubation time, the incubation temperature, and the concentration of the anabiotic can affect the results

The minimal inhibitory concentration test- this method determines the lowest concentration of an antibiotic that can inhibit the growth of a test organism

To rapidly test isolated colonies for the prediction of beta lactamase; a positive beta-lactamase test will show yellow around the disc, A negative beta -lactamase test will show an orange/pink result

Mueller Hinton agar